mouse anti ido1  (Proteintech)

Journal: CNS neuroscience & therapeutics

Article Title: Alleviation of Microglia Mediating Hippocampal Neuron Impairments and Depression-Related Behaviors by Urolithin B via the SIRT1-FOXO1 Pathway.

doi: 10.1111/cns.70379

Figure Lengend Snippet: FIGURE 2 | UB treatment ameliorates depression-like behaviors and neuroinflammation and abnormal expression of SIRT1 and FOXO1 in the hippocampus of LPS-exposed mice. (A) Scheme of the experimental procedure. (B) SPT, FST, and locomotor tests. (C) Relative mRNA levels of TNF-ɑ, IL-1ɑ, IL-1β, IL-6, and IDO in the hippocampus. mRNA levels of M1 type markers iNOS and CD86 (D), and M2 type markers ARG1 and CD206 (E) in the hippocampus. (F and G) Western blot was performed to evaluate the effect of UB on the protein levels of Iba1, IDO, SIRT1, and FOXO1 in the hippocampus. n = 6–7 per group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the Vehicle + Vehicle group or LPS + Vehicle group.

Article Snippet: The primary antibodies employed in this study were as follows: rabbit anti- SIRT1 (Sigma- Aldrich, 07- 131), rabbit anti- FOXO1 (CST, 2880S), mouse anti- β- actin (CST, 3700S), rabbit anti- P65 (CST, 8242T), rabbit anti- phospho- P65 (CST, 3031S), ARG1 (BOSTER, A01106), CD206 (BOSTER, A02285- 2), CD86 (BOSTER, A00220- 4), iNOS (BOSTER, BA0362), ERK (CST, 4695), p- ERK (CST, 4370), rabbit anti- P38 (CST, 9212S), phospho- P38 (CST, 9215), JNK (CST, 9251), phospho- JNK (CST, 9252), rabbit anti- AMPK (CST, 5831S), rabbit anti- phospho- AMPK (CST, 2535S), rabbit anti- Bax (CST, 5023T), mouse anti- Bcl- 2 (CST, 15071T), mouse anti- IDO (Proteintech, 66528- 1- Ig), rabbit anti- Ac- FOXO1 (Invitrogen, PA5- 104560), rabbit anti- Ac- P65 (Abcam, AB19870).

Techniques: Expressing, Western Blot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Persistent tailoring of MSC activation through genetic priming

doi: 10.1016/j.omtm.2024.101316

Figure Lengend Snippet: Genetic priming MSCS via overexpression of IRF1 (A) IRF1 lentiviral construct design. (B) Flow-based quantification of lentiviral transduction efficiency. Images were created using BioRender. (C) IRF1 transgene expression and IFN-γ stimulation at 50 ng/mL for 24 h both yield nuclear localization of IRF1. IRF1 was visualized by immunocytochemical staining. Scale bar, 100 μm. (D and E) IRF1 overexpression and IFN-γ stimulation (50 ng/mL for 24 h) both drive IDO1 transcription (D) and upregulate kynurenine production (E). Relative expression, 2 ΔΔCt , in RT-qPCR analyses was calculated relative to unprimed MSCs for both MSC IFN-γ and MSC IRF1 . Statistical analyses were performed using multiple unpaired t tests. Kynurenine production was assayed using an Ehrlich reaction and analyzed using one-way ANOVA. ∗ p < 0.05. Error bars represent the SD.

Article Snippet: Cells were trypsinized, fixed using BD Cytoperm/Cytofix (BD, Franklin Lakes, NJ, USA), permeabilized with BD Perm/Wash Buffer, and stained at room temperature for 1 h using a PE-labeled mouse anti-IDO1 antibody (12-9477-42, Thermo Fisher Scientific) or a BV786-labeled mouse anti-human HLA-DR antibody (564041, BD) according to the manufacturer’s instructions.

Techniques: Over Expression, Construct, Transduction, Expressing, Staining, Quantitative RT-PCR

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Persistent tailoring of MSC activation through genetic priming

doi: 10.1016/j.omtm.2024.101316

Figure Lengend Snippet: CM from primary MSC IFN-γ and MSC IRF1 abrogate T cell proliferation and activation (A) IDO1 expression measured by flow cytometry shows IRF1 expression results in nearly as much IDO1 expression as IFN-γ stimulation. (B) Kynurenine production, an indicator of MSC activity, was high and indistinguishable between MSC IFN-γ and MSCIRF1. (C) MSC IFN-γ abrogated T cell proliferation slightly more than MSC IRF1 . Severe proliferation of more than 3 generations was abrogated by both MSC IFN-γ and MSC IRF1 . (D) Summary graph of (C). (E) MSC IFN-γ and MSC IRF1 were similar in their ability to minimize CD8 T cell activation via IFN-γ expression. (F) MSC IFN-γ and MSC IRF1 were similar in their ability to minimize CD4 T cell activation via TNF-α expression. One way ANOVA. ∗ p < 0.05, ∗∗∗∗ p < 0.0001. ns, not significant. Error bars represent the SD.

Article Snippet: Cells were trypsinized, fixed using BD Cytoperm/Cytofix (BD, Franklin Lakes, NJ, USA), permeabilized with BD Perm/Wash Buffer, and stained at room temperature for 1 h using a PE-labeled mouse anti-IDO1 antibody (12-9477-42, Thermo Fisher Scientific) or a BV786-labeled mouse anti-human HLA-DR antibody (564041, BD) according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Flow Cytometry, Activity Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Persistent tailoring of MSC activation through genetic priming

doi: 10.1016/j.omtm.2024.101316

Figure Lengend Snippet: Persistence of IRF1 mediated MSC activation (A) Experimental scheme for MSC transduction and transient treatment with IFN-γ and/or budesonide. (B) IDO1 expression responses in non-engineered MSCs that were primed biochemically with 50 ng/mL IFN-γ and/or with 1 μg/mL budesonide (BUD) for 24 h (C) The effect of IFN-γ and/or budesonide on the activation and persistence of IRF1-transduced MSCs. Percent positive IDO1 cells were determined by flow cytometry. Error bars represent the SD, n = 3.

Article Snippet: Cells were trypsinized, fixed using BD Cytoperm/Cytofix (BD, Franklin Lakes, NJ, USA), permeabilized with BD Perm/Wash Buffer, and stained at room temperature for 1 h using a PE-labeled mouse anti-IDO1 antibody (12-9477-42, Thermo Fisher Scientific) or a BV786-labeled mouse anti-human HLA-DR antibody (564041, BD) according to the manufacturer’s instructions.

Techniques: Activation Assay, Transduction, Expressing, Flow Cytometry